Aspergillustubingensis (strain OY907) was isolated from the Mediterranean marine sponge Ircinia variabilis. Extracellular extracts produced by this strain were found to inhibit the growth of several fungi. Among the secreted extract components, a novel anhydride metabolite, tubingenoic anhydride A (1) as well as the known 2-carboxymethyl-3-hexylmaleic acid anhydride, asperic acid, and campyrone A and C were purified and their structure elucidated. Compound 1 and 2-carboxymethyl-3-hexylmaleic acid anhydride inhibited Neurospora crassa growth  and affected hyphal morphology. We produced a N. crassa mutant exhibiting tolerance to 1 and found that a yet-uncharacterized gene, designated mas-1, whose product is a cytosolic protein, confers sensitivity to this compound. The ∆mas-1 strain showed increased tolerance to sublethal concentrations of the chitin synthase inhibitor polyoxin D, when compared to the wild type. In addition, the expression of chitin synthase genes was highly elevated in the ∆mas-1 strain, suggesting the gene product is involved in cell wall biosynthesis and the novel anhydride interferes with its function.The fungus  Although the nature of the associations between sponges and fungi is far from understood, there is accumulating evidence that some sponge-associated fungi have adopted the ability to produce chemical compounds which are structurally diverged from their terrestrial counterparts. These compounds are considered a part of the chemical arsenal which enables niche specialization. In addition, a growing number of studies have indicated that sponge-derived fungi can provide sources of novel bioactive secondary metabolites, exhibiting anticancer, antibacterial, antiviral, anti-inflammatory, antifouling and antifungal agent properties [Aspergillus spp. as producers of bioactive metabolites [A. sydowii isolated from Spongia obscura [A. ustus, isolated from the marine sponge Suberites domuncula that was found to produce seven new drimane sesquiterpenoids which showed cytotoxic activity against a panel of tumor cell lines [A. niger derived from a Caribbean sponge Hyrtios proteus. Chu et al. [l-Trp-l-Phe) produced by the fungus A.versicolor associated with the South China Sea sponge Holoxea sp. and Cohen et al. [Psammocinia sp.-associated A. insuetus. Although many studies describe novel compounds produced by sponge-associated fungi, the understanding concerning the biological activity and the mode of action of those metabolites is, in many cases, lacking. This is especially so in the case of compounds exhibiting antifungal properties.An increasing number of reports on the association of fungi with sessile marine animals is accumulating . Marine operties ,9. Seconoperties , terpeneoperties , peptideoperties , alkaloioperties , nonribooperties  and polyoperties . Many stabolites . Example obscura  and A. ull lines . Varoglull lines  isolatedu et al.  describen et al.  describeN-acetylglucosamine and it is synthesized by chitin synthases. The bulk of the fungal cell wall is comprised of β-1,3 glucan and is synthesized by glucan synthases. The requirement of a functional cell wall for survival, growth, development and pathogenicity of fungal species makes it an attractive target for antifungals, especially due to the fact that some of the constituents of the fungal cell wall are not present in potential hosts [The fungal cell wall is comprised of a mix of cross-linked fibers (mainly the polysaccharides glucan and chitin) and matrix components, primarily proteins and mannans. In filamentous fungi, growth and cell wall assembly occur mainly at hyphal apices, where the carbohydrate polymers are synthesized by membrane-associated enzymes, some of which are transported within vesicles to their site of activity. The polymers are then cross-linked and modified by extracellular proteins . The maial hosts . Exampleal hosts ,24 as weal hosts ,26 and lal hosts  which bl1, produced by an A. tubingensis strain isolated from the sponge Ircinia variabilis. This metabolite exhibits anti-fungal activity, resulting in the impairment of cell wall integrity. Furthermore, we have determined that sensitivity to this compound can be conferred by a single novel protein in Neurospora crassa designated MAS1. Additional metabolites, the known 2-carboxymethyl-3-hexylmaleic acid anhydride [In this study, we report on a novel metabolite, nhydride , and camnhydride , were puet al. [Eurotiales (Aspergillus and Penicillium spp.) associated with the marine sponge Ircinia variabilis. We further analyzed strain OY907 and based on partial sequence of the β-tubulin gene used for identification of Aspergillus species [Aspergillus tubingensis (Accession number KM079500).Paz et al. , identif species , we haveAspergillus species are widespread among marine sponges and usually constitute a large number of the total fungi isolated from many of these organisms [et al. [Aspergillus and Penicillium. The high occurrence of these genera was also evident in the report by Paz et al. [Aspergillus and Penicillium spp. Similarly, among culturable fungi from two South China Sea sponges (Clathrina luteoculcitella and Holoxea sp.), these genera were also highly prevalent [Aspergillus has been widely investigated, to the best of our knowledge this is the first report of an A.tubingensis that has been isolated from a marine environment.rganisms . Holler  [et al.  found thz et al.  stating revalent . AlthougA. tubingensis strain studied here (OY907) was found to secrete chemicals that inhibited the growth of three test fungi: Alternaria alternata, Rhizoctonia solani and Neurospora crassa, as determined by dual culture diffusion assays (inhibition zones wider than 15 mm from the source in the assay Petri dish). The former are Ascomycete and Basidiomycete plant pathogens, respectively, and the latter is a model Ascomycete which is highly amenable to genetic analysis and manipulation. The specific N. crassa. When grown in the presence of 2 mg/mL of the crude extract, hyphae of N. crassa exhibited swollen tips with multiple foci of cytoplasmic leakage. Furthermore, significant amounts of amorphous material accumulated along the hyphae . As the outcome of exposure to the extract could involve more than one compound, fractionation and subsequent structure elucidation of antifungal compounds produced by this strain were carried out.A similar phenotype was observed in 1; m/z 195.1024 corresponding to the molecular formula C11H16O3 and four degrees of unsaturation. Its 1H NMR spectrum (CDCl3 + CD3OD) presented two singlet protons at 6.34 and 5.72 ppm, a triplet proton at 3.40 ppm, two protons at 1.83 and 1.62 ppm, a 8H-multiplet at 1.24 ppm and a triplet methyl at 0.82 ppm. Based on the HSQC experiment, the 13C NMR spectrum of 1 presented four sp2 carbons , a methine carbon (46.6 ppm), five methylene carbons  and a methyl carbon (13.8 ppm). Correlations from the COSY experiment allowed the connection of the methine H-3 (3.40 ppm) with the protons of the diastereotopic methylene-1′ (1.82 and 1.61 ppm) and of the later with the eight-proton multiplet at 1.24 ppm. This multiplet was further correlated with the triplet methyl suggesting that a normal hexyl was attached to methine-3. This was supported by the HMBC correlations summarized in 3J) HMBC correlations with the carbon resonating at 168.7 ppm (C-5). The terminal methylene protons exhibited additional HMBC correlations (2J) with a quaternary olefin carbon , strong correlations (3J) with C-3 and weak correlations (4J) with the sp2 carbon at (176.0 ppm). H-3 exhibited correlations with all four sp2 carbons and two methylene carbons resonating at 30.8 (1′) and 27.3 ppm suggesting that it was bridging between the aliphatic and unsaturated moieties of 1. The diastereotopic protons of methylene-1′ exhibited HMBC correlations with the two quaternary sp2 carbons resonating at 176.0 and 138.5 ppm, with methine-3 and with two methylene carbons resonating at 27.3 and 29.0 ppm. These correlations established the attachment of the hexyl moiety to methine-3, the vicinity of methylene-1′ to the carboxyl carbon resonating at 176.0 ppm and to the quaternary olefin carbon (138.5 ppm), and assigned the methylene resonating at 27.3 and 29.0 ppm as 2′ and 3′, respectively. The correlations of the methyl protons with the methylene carbons resonating at 31.4 and 22.4 ppm, assigned them as C-4′ and C-5′, respectively. Based on the molecular formula inferred from the HR mass measurements the two carboxyls of 1 are engaged in an anhydride linkage. Based on the arguments summarized above the structure of tubingenoic anhydride A (1) was established as 3-hexyl-4-methylenedihydrofuran-2,5-dione.Tubingenoic anhydride A 1;  was isol1) is a polyketide metabolite exhibiting anti-fungal activity (N. crassa MIC = 330 μM) derived from the conjugation of octanoic acid and C4 metabolite, probably oxaloacetic acid and decarboxylation. Related metabolites were previously isolated from several Aspergillus spp. 2-Carboxymethyl-3-hexylmaleic acid anhydride was isolated from Aspergillusawamori as a weak antibiotic of gram-positive bacteria [N. crassa (MIC = 207 μM). 2-Carboxymethyl-3-hexylmaleic acid anhydride and 2-methylene-3-hexylbutanedioic acid were isolated from A. niger as enhancers of seed germination, and shoot and root growth in cauliflower seedlings [Tubingenoic anhydride A  resistance cassette. The NatR transformants were examined for resistance to the antifungal compounds. Analysis of progeny from purified homokaryons crossed to the wild type confirmed the genetic link between NatR and the ability to grow on the crude extract. One of the transformants (m-42) was used for further analysis. To determine the integration site of the pNV15 construct, DNA contiguous with the inserted plasmid was retrieved by a plasmid rescue procedure. Genomic DNA of the resistant mutant was digested with BamHI (which does not cleave within the mutagenesis plasmid) and the ligated product was sequenced. DNA sequence flanking the pNV15 insert was then used to probe the N. crassa genome database [mas-1 (Marine-Aspergillus Sensitive 1). Even though the genome of several hundred fungal strains has been sequenced, many genes in the highly diverse members of this kingdom have not been assigned functions. This is also true for N. crassa, the first published full genome of a filamentous fungus [In order to provide insight on the mechanism(s) involved in conferring sensitivity of database  The disrs fungus ,38, whermas-1 is a unique, yet uncharacterized gene, several measures were taken in order to fully confirm the relevance of mas-1 to the sensitivity of N. crassa to the A. tubingensis metabolites. To verify that no annotation error  had occurred during the analysis of the N. crassa genome, we examined the ∆mas-1 (NCU03140) deletion strain obtained from the FGSC and compared it to strain m-42. As both strains were resistant to the antifungal extract, we concluded that mas-1 is, indeed, involved in conferring the biological activity of the A. tubingensis metabolites to N. crassa (1), the wild type was highly sensitive to the metabolite compared to the ∆mas-1 strain, at a concentration of 0.8 mg/mL. As . crassa B. In add∆mas-1 strain were placed on medium amended with the A. tubingensis extract, they germinated to produce normal appearing hyphae, with the exception of a few cases of hyphal swelling and cytoplasmic leakage. Hence, even though the absence of mas-1 confers a high degree of tolerance to the crude extract, at concentrations of 2 mg/mL or higher, some effects of the antifungal compound could still be observed .When conidia of the observed , indicatmas-1 is a small gene and is closely linked to two additional genes: Histidine biosynthesis trifunctional protein  and Lysophospholipase (NCU03141)   A, that mgration) . Taken tA. tubingensis extract indicated that hyphal integrity may be compromised, we analyzed the sensitivity of the ∆mas-1 strain to several fungicides whose function results in impaired hyphal integrity. Sublethal concentrations of Fluconazole (an ergosterol biosynthesis inhibitor) or fludioxonil (an activator of the hyperosmotic stress response pathway) did not differentially affect the growth of mas-1 when compared to the wild type . These data suggest that the chitin constituent of the cell wall may be compromised in a ∆mas-1 background. Furthermore, the fact that the increased tolerance of the ∆mas-1 strain to Polyoxin D was not accompanied by a similar trend when exposed to the glucan synthesis inhibitor caspofungin, suggests that the functional link of MAS1 to chitin biosynthesis is specific. Alternatively, the lack of MAS1 may also affect chitin synthesis in an indirect manner, e.g., via altering the capacity of the fungus to uptake the chitin synthase inhibitor. Regardless of the precise mechanism involved, the interest in all aspects of fungal cell wall integrity as a potential target for antifungals [mas-1  play in maintenance of fungal cell integrity. The cross-species sensitivity and the similar defects in the test fungi analyzed on the one hand, and the fact that mas-1 is a unique gene, on the other, suggest that other genes in A. alternata and R. solani (and likely in N. crassa as well) are involved in conferring sensitivity/tolerance to the newly identified compound. Analysis of addition N. crassa mutants tolerant to the compound as well as further analysis of the other test fungi may provide a better mechanistic understanding of the compounds’ mode of action. Such information has the potential to be utilized in the development of antifungal treatments based on cell wall impairment as a target.As the nature of the phenotypic consequences in the presence of the ild type . Howeverifungals  warrantsA. tubingensis antifungal products and cell wall integrity, we examined the relative expression of the seven chs genes in the wild type and in ∆mas-1. When exposed to a sublethal concentration (1 mg/mL) of the A. tubingensis crude extract, a significant increase in the transcript levels of all chs genes was observed in the wild type strain . These higher expression levels include chs-4, a gene whose product has been implied to be an “auxiliary enzyme” under stressful conditions [mas-1 confers a precondition in which the fungal cell has an elevated pool of chitin synthases that contributes to its tolerance to the A. tubingensis products. Even though exposure to sublethal A. tubingensis extract concentrations results in increased chs expression in the wild type, the expression levels may not be sufficient to confer tolerance once the fungus has been exposed to the antifungal compounds. It is also possible that MAS1 functions in additional manners that increase tolerance, such as an effect on chitin synthase localization/activation or via additional components involved in cell wall integrity. Overall, these results indicate that the A. tubingensis crude extract has an effect on chs genes expression and that at least part of the changes in chs genes expression is mediated by MAS1. To provide additional insight as to the link between the e strain , an expenditions . It is tN. crassa inducible ccg-1 promoter pMF272 [mas-1 strain was co-transformed with pLK4 and pNV15 (conferring NATR). The resulting transformants restored sensitivity to the crude extract, verifying the functionality of the MAS1::GFP fusion insert. Western blot analysis using anti-GFP antibodies was used to verify the presence of tagged MAS1. Confocal microscopy indicated that MAS1::GFP is distributed throughout the hyphal cell in a punctate manner, with marked presence of the protein in yet unidentified round organelles  in specific cellular compartments. Furthermore, MAS1 was not detected in conidia. Though some chitin synthases are ferried in distinct vesicles [In order to determine the cellular localization of MAS1, we constructed a MAS1::GFP fusion (designated pLK4) driven by the r pMF272 . The ∆maganelles . These bvesicles , the patvesicles . N. crassa provides an attractive platform for the analysis of the mode of action of novel compounds as a step in the development of antifungal strategies.In summary, marine sponge-associated fungi have a great potential to be a rich source of promising antifungal compounds, and this may be due, in part, to the unique ecological grow conditions . Even though the compounds described have relatively high MICs, the potential of combining these or structurally related compounds with more effective antifungals  may provide an additional basis for antifungal intervention. The abundance of resources available for the analysis of 1H and 125.76 MHz for 13C. COSY-45, gTOCSY, gROESY, gHSQC and gHMBC spectra were recorded using standard Bruker pulse sequences. HPLC separations were performed on a Jasco HPLC system  and on an Agilent 1100 series HPLC system. Optical rotation values were obtained on a Jasco P-1010 polarimeter at the sodium D line (589 nm). UV spectra were recorded on an Agilent 8453 spectrophotometer. High-resolution MS were recorded on a Waters MALDI Synapt instrument. NMR spectra were recorded, at 25 °C, on a Bruker DRX-500 spectrometer at 500.13 MHz for Aspergillus sp. strain (OY907) previously isolated from the Mediterranean sponge Ircinia variabilis  and Rhizoctonia solani (TP6) form the Department of Plant Pathology and Microbiology collection were maintained on PDA. N. crassa strains used in this study were the wild type (74-OR23-1A), a Δmas-1N. crassa knock out mutant strain , as well as chs-1, -3,-4,-5,-6 and 7, which were obtained from the Fungal Genetic Stock Center. The chs-2RIP strain was described in a previous study [N. crassa strains were grown in either liquid or solid (supplemented with 1.5% agar) Vogel’s minimal medium supplemented with 1.5% (w/v) sucrose (Vs). Unless stated otherwise, all growth and manipulations of N. crassa were as previously described [d-glucan synthase inhibitor  and polyoxin D  a chitin synthase inhibitor. For growth rate measurements, 10 μL of a conidial suspension (1 × 107 conidia/mL) were inoculated on Petri dishes or race tubes containing Vs and growth was measured periodically.An z et al. ) was culus study . N. crasescribed . When re N. crassa, A. alternata and R. solani [N. crassa and the other two fungi, respectively), the effect of diffused compounds from the marine isolate was assessed. The formation of inhibition zones wider than 15 mm between the OY907 source and the test organisms was considered as an indicator for the presence of inhibitory compounds. To determine the presence of secreted anti-fungal compounds, an agar medium diffusion assay was performed using. solani . A mycel. The mycelium was separated from the growth medium by filtration and then extracted two times with ethyl acetate 1:1 (v:v). Ethyl acetate extracts were combined, filtered and evaporated. The dry crude extract was dissolved in dimethylsulfoxide (DMSO) and tested for its antifungal activity. The extract was fractionated on a reversed-phase open column followed by gel-filtration on Sephadex LH-20. Each fraction was tested for biological activity by amending the N. crassa growth medium with a range of concentrations (0.5–2 mg/mL) of the dried fraction. Final purification of the active  compounds was achieved by HPLC separation. The active fraction was retested for antifungal activity and the structure of the purified compound was elucidated by1D and 2D NMR technique and HR mass spectrometry.Strain OY907 was mass cultivated on PDB in stationary flasks for 25 days at 27 °C in the dark1).  −6.3 ; UV (MeOH) λmax (log ɛ) 202 (3.58); IR (CHCl3) νmax 3020, 2929, 2858, 1708, 1629, 1522,1421,1212 cm−1; For NMR data see m/z 195.1024 .Tubingenoic anhydride A  in a 100 µL reaction volume. The ligated products were then cloned into E. coli HIT DH5α competent cells (RBC Bioscience) and resulting clones were sequenced.In order to study the function of the poration . We usedporation  that linrocedure . Genomicmas-1 into the m42 mutant, the mas-1 gene was amplified by using primers f-1200 and r-609 that include the mas-1 gene as well as 1200 bp and 609 bp of DNA upstream and downstream of the coding region, respectively. The PCR product was cloned into a pDRIVE expression vector (QIAGEN) according to the manufacturer’s instructions and designated pLK3. pLK3 was restricted with StuI and the linear construct was cotransformed into the N. crassa m-42 strain along with pCSN44 (which contains a hygromycin B resistance cassette) [A. tubingensis growth medium. PCR amplification of the inserted cassette utilizing primers hphf and hphr and primers mas-1f and mas-1r (For introducing a wild-type copy of assette) . The resd mas-1r , were usN. crassa has been shown to have seven genes encoding chitin synthases [mas-1 conferred changes in the expression of the different chitin synthase (chs) genes, we preformed Real Time PCR for all the seven chs genes. Since genes encoding for CHS in N. crassa are highly similar, manual primer design was carried out in order to identify unique 80–150 bp amplicons. Confirmation of primer set specificity was performed by Real Time PCR on cDNA templates of the mutants lacking genes encoding for CHS.ynthases . In ordechs gene expression, RNA was extracted from conidial germlings (from the wild type and ∆mas-1 strains) that were grown on liquid Vs amended with 1 mg/mL  of the crude extract for 4 h at 34 °C at 150 rpm. The RNA samples were purified with the RNeasy Plant Mini Kit (Qiagen) as previously described [chs genes was performed on an ABI StepOnePlus Real-Time PCR Sequence Detection System and software (Applied Biosystems) as described [chs 1-7 primers (see beta tubulin gene (NCU04173.3). In all experiments, samples were amplified in triplicate, and the average cycle threshold was then calculated and used to determine the relative expression of each gene. Relative expression levels were calculated using the 2−Δct method [For determining escribed . Purifieescribed . RT-PCR mers see . Total ct method .in vivo for cellular localization, MAS1 was tagged with a green fluorescent protein (GFP). The putative mas-1 ORF (381 bp) was amplified, without the stop codon, as an XmaI/XbaI fragment (390 bp) utilizing primers gfp-1f and gfp-1r  [mas-1 strain. pLK2 was digested with SapI/PstI and the gel-purified fragment (4 kb) was used to cotransform the mutant strain along with pNV15. The transformed isolates were selected for their ability to grow on NAT. PCR amplification and subsequent sequencing of pLK2 utilizing primers gfp-2f and gfp-2r   which isY598428) . This yid gfp-2r , were ped nat-1r  were useIrcinia variabilis as Aspergillustubingensis. We have purified and identified a novel compound that exhibits antifungal properties and have identified it as Tubingenoic anhydride A. We have determined that a small (91 aa), yet uncharacterized protein, MAS1, confers sensitivity to the antifungal products in N. crassa. The Δmas-1 strain was two-fold more resistant to the chitin synthase inhibitor Polyoxin D than the wild type, suggesting, that the mas-1 gene is involved in the cell wall integrity. Moreover, the constitutive elevated expression of chitin synthase genes in the Δmas-1 mutant supports the link of MAS1 to cell wall integrity. In this study, we have identified a fungal strain previously isolated from the Mediterranean marine sponge


SNDR) and an 87-dB dynamic range when integrated over a 10-kHz bandwidth. The total power consumption of the modulator is 13 μW, which corresponds to a figure-of-merit (FOM) of 45 fJ/conversion step. These competitive circuit specifications make this design a good candidate for building high precision neurosensors.Recorded neural data are frequently corrupted by large amplitude artifacts that are triggered by a variety of sources, such as subject movements, organ motions, electromagnetic interferences and discharges at the electrode surface. To prevent the system from saturating and the electronics from malfunctioning due to these large artifacts, a wide dynamic range for data acquisition is demanded, which is quite challenging to achieve and would require excessive circuit area and power for implementation. In this paper, we present a high performance Delta-Sigma modulator along with several design techniques and enabling blocks to reduce circuit area and power. The modulator was fabricated in a 0.18-μm CMOS process. Powered by a 1.0-V supply, the chip can achieve an 85-dB peak signal-to-noise-and-distortion ratio ( Growing concerns for human health have stimulated the development of biomedical devices ,15,16,17FOM. (1) We have used high density metal-oxide-semiconductor (MOS) capacitors to replace metal-insulator-metal (MIM) capacitors for reducing circuit area [In this paper, we present a Delta-Sigma ADC for building high precision recording microsystems. Compared to recent Delta-Sigma ADC designs ,27,28,29uit area . As detauit area , our desuit area .To implement the proposed MBSO-based architecture, several circuit techniques have been incorporated as summarized below. First, we have performed careful characterization with each type of MOS capacitor. Based on the simulation results, we have chosen the optimal type of capacitor for each integrator. Second, a switched op-amp technique has been used where amplifiers are automatically turned off when they are not in use. Realizing such high performance switched op-amp depends on the bias circuits. In this design, novel bias circuits have been proposed to reduce switching power loss. Third, the static power of the quantizer has been removed through an elegant circuit arrangement. Finally, a new half-cycle operating resonator scheme has been designed for reducing in-band quantization noise. These techniques together boost the performance of the modulator and make it a competitive candidate for building high precision recording microsystems.in vivo preparations and epilepsy patients. The rest of this paper is organized as follows. In vivo neural data recorded from extracellular space consist of both LFPs and extracellular spikes. The amplitude of extracellular spikes is inversely proportional to the distance between the recording site and neuron [d neuron . Neural d neuron . Howeverin vivo preparations sampled at 40 kHz; (2) full-spectrum recordings from epilepsy patients sampled at 32 kHz. To study the required system dynamic range to accommodate both neural activities and artifacts, we have analyzed data sequences obtained from different setups: (1) ENOB) of dynamic range in vivo data sequences are shown in in vivo preparations and epilepsy patients are (12.06–13.58)-bit and (11.73–13.09)-bit, respectively.The formula to estimate the data dynamic range is shown below:The proposed Delta-Sigma modulator topology is shown in SR), gain bandwidth (GBW), voltage gain, nonlinearity, charge injection, This section gives the detailed analyses of the proposed modulator. A number of circuit design issues, including slew rate (NTF) of the proposed modulator can be calculated as:The noise transfer function  is set to be equal and scanned from 0 dB–120 dB. For the dotted line with “*”, the voltage gains of the first two stage OTAs are scanned from 0 dB–120 dB, while the gains of the third and fourth stage OTAs are fixed at 60 dB. For the dotted curve with “∘”, the voltage gains of the third- and fourth-stage OTAs are scanned from 0 dB–120 dB, while the gains in the first two stages are fixed at 60 dB. According to the simulation results, the OTA gains in the first two stages can be reduced to 40 dB, while the OTA gains in the third and fourth stages should be more than 60 dB.SR, finite GBW, etc. As shown in rly used ,46,47. IA pseudo DWA behavioral block as shown in SNDR performance over the required bandwidth.Figure Considering efficiency in terms of area, MOS capacitors are preferred due to their higher capacitance density . Figure For circuit analysis, 2) at a moderate bias voltage range (0.4 V–1.4 V) and poor linearity (<4.1%). The parallel-PMOS type gives a moderate density (2.35 fF/μm2) at a narrow bias voltage range (±0.2 V) and moderate linearity (<3.5%). The series-PMOS type has a moderate density (2.05 fF/μm2) with a wide bias voltage range (0.2 V–1.4 V) and good linearity (<2.8%). The density of the MIM capacitor is only 0.97 fF/μm2, though it allows a wide bias voltage range (−0.4 V–1.4 V) and good linearity (<0.1%).A, To reduce circuit power and noise, it is important to select an appropriate switched op-amp for each integrator. The simulation results of both NMOS input-pair switched op-amp and PMOS input-pair switched op-amp are shown in I is the reference current, W and L are the transistor width and length; The drain current , the random offset with a standard deviation of up to 0.4 LSB  can be easily accommodated in the proposed modulator.A local resonator scheme with a coefficient of 1/50 is applied to reduce in-band quantization noise power. The traditional resonator as shown in SQNR, a novel area- and power-efficient resonator scheme using a parallel-PMOS capacitor is proposed to realize a small coefficient of 1/50. Compared to a modulator using only an MIM capacitor, 55% capacitor area is saved in the proposed modulator design.SNDRversus different input amplitudes. For Modulator A, the measured peak SNDR and dynamic range are 80 dB and 87 dB, respectively, while the total power consumption is 20 μW with an FOM of 122 fJ/conversion step. For Modulator B, the measured peak SNDR and dynamic range are 85 dB and 87 dB, respectively, while the total power consumption is 13 μW. These specifications correspond to an FOM of 45 fJ/conversion step. SNDR at different signal frequencies and power supplies. The proposed modulator was fabricated in a 0.18-μm CMOS process. The chip micrograph and circuit testing setup are shown in in vivo preparations and epileptic patients suggests that a wide system dynamic range is required to simultaneously record both neural activities and artifacts. To meet the required dynamic range, yet low power operation, this paper presents design analyses, circuit implementation and measurement of a Delta-Sigma modulator chip. Powered by a 1.0-V supply, the chip can achieve an 85-dB peak SNDR and an 87-dB dynamic range when integrated over a 10-kHz bandwidth. The total power consumption of the modulator is 13 μW, which corresponds to an FOM of 45 fJ/conversion step. The competitive circuit specifications make this design a good candidate for building high precision neurosensors.Data analysis on recorded sequences from both


The Pediatric Quality of Life Inventory™ Generic Core Scales (PedsQL™) 4.0 is a generalized assessment of health-related quality of life (HRQoL) based on adolescent self-report and parent proxy-report. This study aims to determine the construct validity and reliability of PedsQL™ 4.0 among a sample of Malaysian adolescents and parents.A cross-sectional study was carried out at three selected public schools in the state of Selangor. A total of 379 Malaysian adolescents completed the PedsQL™ 4.0 adolescent self-report and 218 (55.9%) parents completed the PedsQL™ 4.0 parent proxy-report. Weight and height of adolescents were measured and BMI-for-age by sex was used to determine their body weight status.2 values. Cronbach’s alpha coefficients of the adolescent self-report (α = 0.862) and parent proxy-report (α = 0.922) showed these instruments are reliable. Parents perceived the HRQoL of adolescents was poorer compared to the perception of the adolescent themselves . There was no significant difference in total HRQoL score between male and female adolescents . Parent proxy-report was negatively associated with the adolescents’ BMI-for-age  whereas no significant association was found between adolescent self-report and BMI-for-age .There were 50.8% male and 49.2% female adolescents who participated in this study (14.25 ± 1.23 years). The prevalence of overweight and obesity (25.8%) was four times higher than the prevalence of severe thinness and thinness (6.1%). Construct validity was analyzed using Confirmatory Factor Analysis (CFA). Based on CFA, adolescent self-report and parent proxy-report met the criteria of convergent validity  > 0.5, Construct Reliability > 0.7) and showed good fit to the data. The adolescent self-report and parent proxy-report exhibited discriminant validity as the AVE values were larger than the RAdolescent self-report and parent proxy-report of the PedsQL™ 4.0 are valid and reliable to assess HRQoL of Malaysian adolescents. Future studies are recommended to use both adolescent self-report and parent-proxy report of HRQoL as adolescents and parents can provide different perspectives on HRQoL of adolescents. The WHO Quality of Life Group  defines The physical dimension of HRQoL measures the changes in physical activities carried out by individuals daily. The emotional dimension of HRQoL assesses the satisfaction, achievement of personal goals, personal control, social interaction, self-concept and self esteem , whereasThere is a need to assess the HRQoL in a general population as poor HRQoL may not only be caused by the presence of a disease, but could also be due to factors such as stigmatization , physicaThe PedsQL™ 4.0 was previously used to measure the HRQoL of thalassaemia  and disaThis cross-sectional study was conducted from 25 June to 19 July 2011 among secondary school students in the districts of Kajang and Bangi, state of Selangor, Malaysia. Three schools which met the inclusion criteria of being co-educational, multiracial in composition, non-religious and government public schools were selected for this study. Students in the examination school year (Form 3 and Form 5) were excluded from this study as instructed by the Ministry of Education.Ethics approval were obtained from the Medical Research Ethics Committee of the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM/FPSK/PADS/T7-MJKEtikaPer/F01 (JPD(U)_SEPT (10)38). Also, approval to conduct the study in government public schools was obtained from the Ministry of Education Malaysia, Selangor State Department of Education and the schools involved. Four trained researchers were involved during data collection. Their training focused on techniques of conducting anthropometric measurements and providing a standardized flow of instructions and procedures to the adolescents involved in the study. Any question by the adolescents on the questionnaire was answered by the project leader. Information sheets on the study and consent forms were distributed to the adolescents and their parents prior to data collection. Signed informed consent forms were collected on the day of data collection. A total of 390 adolescents and their parents were initially invited to participate in this study. There were 379 (97.2%) adolescents who consented and completed the self-report of PedsQL™ 4.0 whereas 218 (55.9%) parents returned the parent proxy-report of PedsQL™ 4.0.Socio-demographic information including sex, ethnicity, age and date of birth were obtained from the adolescents. Education level and monthly income of parents were collected from the parents.Body weight of adolescents was measured using a TANITA Digital Weighing Scale (HD 319) to the nearest 0.1 kg whereas a SECA body meter (SE 206) was used to measure height to the nearest 0.1 cm. Body weight status of adolescents was classified based on BMI-for-age by sex using the WHO Growth Reference  .The PedsQL™ 4.0 version 13-18 years was used in this study. It is a generic tool that assesses the HRQoL of patients or healthy population among children and adolescents .The PedsQL™ 4.0 can be interpreted based on a scale order, lower and higher order. The lower order scale encompasses four dimensions : (1) PhyThe PedsQL™ 4.0 requires the adolescent and parent to recall the frequency of problems which occurred with the adolescent in the past one month. A five-point response Likert scale  was used for all items. All the items were then reverse scored and transformed into a 0-100 point scale , whereby a higher total score of PedsQL™ 4.0 indicates a better HRQoL of the adolescent.The adolescent self-report and parent-proxy report of PedsQ™ 4.0 were translated from English to the Malay language by two postgraduate students with backgrounds in the Health Sciences fluent in both languages. The questionnaire was then back-translated into English by another two bi-lingual postgraduate students also with backgrounds in the Health Sciences. The English back-translated version was consistent with the original PedsQ™ 4.0 adolescent self-report and parent proxy-report.Data were analyzed using SPSS version 19. Missing values were replaced with the mean values of the items of the respective scale per individual respondent. Internal consistency which is defined as the extent to which all items of a test measure the same latent variable  was deteConfirmatory factor analysis (CFA) was carried out using the Analysis of Moment Structure (AMOS) software version 19 to assess construct validity. Construct validity is a set of measured items which reflects the theoretical latent construct those items are designed to measure ,24. In tAs part of convergent validity, the model fit of the instrument was tested. A number of fit indices were used to test the model fit: goodness-of-fit index (GFI), adjusted goodness-of-fit index (AGFI), comparative fit index (CFI), normed fit index (NFI), Tucker Lewis index (TLI) and root mean square error of approximation (RMSEA). Model fit was considered acceptable when the values of GFI, CFI, TLI, AGFI and NFI were above 0.9 and RMSEA less than 0.8 . Any fou2) was used as this indicates the items of each dimension belong to the construct they are associated with rather than the other constructs.Discriminant validity refers to a set of variables presumed to measure different dimensions . DiscrimA total of 379 adolescents , with a mean age of 14.25 ± 1.23 years, from three public schools in the state of Selangor participated in this study  criterion as it is within the acceptable range (Construct Reliability (CR) ≥ 0.7). Specifically, CR of all items in the lower-order scale of the PedsQL™ 4.0 ranged from 0.768 to 0.853 for the adolescent self-report and 0.929 to 0.972 for the parent proxy-report , GFI (>0.9), NFI (>0.9), TLI (>0.9), AGFI (>0.9), RMSEA (<0.8) were used to determine model fit for both lower and higher-order scale of the PedsQL™ 4.0. Of the fit indices mentioned, four fit indices which were within the acceptable range were chosen to determine the model fit of the data . Data ofIn addition, the non-significance of chi-square goodness-of-fit (CMIN/df) demonstrates a good model fit. The recommended value for CMIN/df is less than 5.0 . The CMI2). A larger AVE value when compared to the squared correlation between the dimensions (R2) would provide evidence of discriminant validity. The correlation between the lower and higher-order scale of the PedsQL™ 4.0 adolescent self-report ranged from 0.35 to 0.66. Thus, a maximum R2 value derived from this correlation was 0.44. Table 2 of 0.31. As the AVEs were more than 0.31, the constructs exhibit sufficient discriminant validity for the parent proxy-report of the PedsQL™ 4.0.Further, discriminant validity was determined for the lower and higher-order scales of the PedsQL™ 4.0. In this study, discriminant validity was determined by comparing the AVE values of the dimensions with the squared correlation between the dimensions R. A largeCronbach’s alpha coefficients (α) across the dimensions of the PedsQL™ 4.0 for the adolescent self-report and parent proxy-report are presented in Table Distribution of HRQoL scores in adolescents according to adolescent self-report and parent proxy-report.The descriptive statistics of adolescent HRQoL scores according to adolescent self-report and parent proxy-report are presented in Table When compared by dimensions between the male and female adolescents, there was a significant difference in the emotional and social functioning whereby the mean emotional functioning scores were 69.52 ± 22.63 and 65.27 ± 21.29 for the male and female adolescents respectively  Table . The meaFurther, there was no significant association between BMI-for-age (z-score), HRQoL dimensions and total HRQoL score of the adolescent self-report. On the other hand, there was a significant negative weak relationship between social functioning, psychosocial health dimension and total HRQoL score reported by parents with BMI-for-age (z-score)  respectively , factor loading and CR of more than 0.5. These results are in agreement with previous studies ,20 where2 of the correlations [When taking the results of fit-indices into consideration  to test model fit, the lower and higher-order scales of the PedsQL™ 4.0 showed a range of average to good data fit for both adolescent self-report and parent proxy-report. This result supports the findings from a study by Petersen et al. 2009) who have reported that the PedsQL™ 4.0 met the cut-off criteria of the fit indices indicating an acceptable model fit 09 who ha. Discrimelations . This stelations ,31-34.In this study, parents perceived HRQoL of their children as being poorer than the perception of the adolescents themselves. Parents scored lower in both the lower and higher-order scales of PedsQL™ 4.0 when compared to the scores by the adolescents, indicating parents have a different perspective on their children’s HRQoL. Previous studies also reported that HRQoL scores by parent proxy-reports have been consistently lower than the adolescent self-report ,18. The Although this study reported no difference in overall HRQoL by sex, adolescent males were found to have better emotional functioning than adolescent females. This result is in line with many studies as female adolescents have reported poorer HRQoL, specifically in the physical and emotional functioning dimensions when compared to the male adolescents ,38-40. PThe prevalence of overweight and obesity among adolescents in the district of Kajang and Bangi has almost doubled from 19.5%  to 25.8%Since the overweight and obesity problem is one of the most important factors contributing to poor HRQoL among adolescents, the current study assessed the correlation between BMI-for-age and HRQoL. Parent proxy-report of the PedsQL™ 4.0 showed a significant inverse association between total HRQoL score, social functioning, psychosocial health with BMI-for-age. This indicates that as BMI-for-age of the adolescents’ increases, the total HRQoL score by the parent proxy-report decreases. The contradicting findings between the reports could be due to the different perceptions of HRQoL among the adolescents and their parents ,36. FurtOur study has several limitations that should be taken into consideration. Firstly, the participants involved in this study were 13 to 17 years old, thus this study is not applicable to children or early adolescents. Future studies are recommended to involve young adolescents since the PedsQL™ 4.0 is valid to be used among adolescents (10-19 years) as defined by WHO . SecondlThis study demonstrates that adolescent self-report and parent proxy-report of PedsQL™ 4.0 Generic Core Scales are valid and reliable to assess the HRQoL of Malaysian adolescents in a non–clinical population. Therefore, it would be recommended to use both the adolescent self-report and parent proxy-report of PedsQL™ 4.0 in future studies. The parent proxy-report of PedsQL™ 4.0 is able to provide a different perspective on the HRQoL of their children.


Passive stepping was performed by a robotic gait trainer system (Lokomat®) while a single pulse of electrical stimulation to the median nerve elicited H-reflexes in the FCR. The amplitude of the FCR H-reflex was significantly suppressed during passive stepping. Although 30 min of passive stepping was sufficient to elicit a persistent H-reflex suppression that lasted up to 15 min, 5 min of passive stepping was not. The duration of H-reflex suppression correlated with that of the stepping. These findings suggest that the accumulation of stepping-related afferent feedback from the leg plays a role in generating short-term interlimb plasticity in the circuitry of the FCR H-reflex.Both active and passive rhythmic limb movements reduce the amplitude of spinal cord Hoffmann (H-) reflexes in muscles of moving and distant limbs. This could have clinical utility in remote modulation of the pathologically hyperactive reflexes found in spasticity after stroke or spinal cord injury. However, such clinical translation is currently hampered by a lack of critical information regarding the minimum or effective duration of passive movement needed for modulating spinal cord excitability. We therefore investigated the H-reflex modulation in the  Part of the neuronal coordination between the fore- and hind-limbs observed in quadrupedal locomotion is preserved in humans Dietz, . This coDuring rhythmic arm movement, Hoffmann (H-) reflex amplitude in stationary leg muscles is strongly suppressed  H-reflex was decreased during and after passive leg cycling in the supine position  H-reflex. This previous work signifies that the H-reflex amplitude is strongly affected by the type of cycling movement (active or passive) and the limb position with respect to the limb where the H-reflex was recorded. Interestingly, Javan and Zehr  with normal neurological functions provided informed written consent before participation in the study. The protocol of this project was approved by the local ethics committee of the National Rehabilitation Center for Persons with Disabilities and was conducted in accordance with the guidelines of the Declaration of Helsinki (1964).® is fully programmable, including the control of the knee and hip kinematic trajectories during stepping with body weight loading  was used to produce “passive stepping” (defined as stepping movements driven by the DGO while relaxing the leg muscles). In this DGO stepping, there may be some low-level, involuntary muscle activation . The second assessed the effects of stepping duration  on FCR H-reflex suppression . In each experiment, passive stepping and static standing trials were performed. Experiments 1 and 2 were conducted on separate days. In Experiment 2, three training sessions of 5, 10, or 15 min stepping periods were conducted on the same day. These training sessions were conducted in a randomized order with an intersession interval of approximately 30–40 min to allow H-reflex amplitude to return to the control values.To explore the long-lasting effects of passive stepping on forearm H-reflex amplitudes, the subjects participated in two experiments using the Lokomat system. The first was an investigation of the long-lasting suppression of FCR H-reflex amplitude after 30 min of passive stepping . The second measured maximum M-waves (Mmax) after 30 min of passive stepping (n = 5). The two control experiments were conducted on separate testing days.Additional control experiments were conducted using the Experiment 1 protocol. In the first experiment, H-reflex amplitude was measured after standing on the treadmill for 30 min without passive stepping  of the median nerve with a constant current electrical stimulator  sizes during the static condition on the treadmill  amplitudes were measured while standing  and stepping . At the beginning of Experiments 1 and 2, recruitment curves of FCR H-reflex and M-wave were constructed to determine both the Mextensor carpi radialis (ECR), rectus femoris (RF), biceps femoris (BF), tibialis anterior (TA), and SOL on the right side. EMG signals were obtained with surface electrodes  placed over the muscle belly after decreasing impedance of skin to below 10 kΩ by light abrasion and cleaning with alcohol. All EMG signals were amplified (1000×) and band-pass filtered between 15 Hz and 3 kHz via a bioelectrical signal amplifier system  activity was recorded from the FCR, post hoc test.Peak-to-peak amplitudes of M-waves and H-reflexes during stepping and standing were normalized to the control values of H-reflex amplitudes. These values were compared using the 1-way repeated measurement of analysis of variance (ANOVA). Multiple comparisons were performed using Dunnett’s f index where a small effect was 0.1, medium effect was 0.25, and large effect was 0.40  to measure the effect size was performed on our data set . In contrast, significant correlation coefficients were not obtained when fitted with the other methods (p > 0.05). Sigmoidal curve fitting analyses were performed using Sigmaplot 10 software . Data are described as means ± standard errors (SEM) and significant differences were set at p < 0.05 in all cases. All statistical tests except for the curve fitting analyses were performed using SPSS software . F-values and degrees of freedom were obtained after the Greenhouse-Geisser correction, where appropriate . After 30 min of stepping, reflex amplitudes were significantly suppressed for 15 min . The 1-way repeated ANOVA of H-reflex amplitudes described a main effect for time courses  = 20.898, f = 4.250, p < 0.01). In contrast, we found no significant differences between the M-wave amplitudes in the control and test conditions  = 0.988, f = 0.746, p > 0.05; Figure Figure Experiment 2 was conducted to determine the duration of passive stepping required to induce prolonged suppression of H-reflex amplitudes. In Experiment 1, post-stepping suppression was maintained after 15 min of stepping  = 17.648, f = 1.332, p < 0.001, Dunnett test; p > 0.05 , M-wave: 1-way ANOVA; F = 1.385, f = 0.416, p = 0.269]  = 38.489, f = 1.84, p < 0.001, Dunnett test; p < 0.001 (Pre vs. 0 min after 10 min stepping), M-wave: 1-way ANOVA; F = 1.022, f = 0.33, p = 0.386] whereas after 15 min of stepping, suppression was maintained for up to 3 min  . It is notable that the suppression of the H-reflex gradually tapering off with an increased duration of stepping. In fact, values of the slope parameter [K value, the Boltzman sigmoidal function  = 1.296, f = 0.938, p > 0.05; M-wave: F = 0.212, f = 0.420, p > 0.05). In Mmax experiment after 30 min passive stepping, also, a significant modulation of the Mmax amplitude was not detected after 30 min of passive stepping  = 1.295, f = 0.468, p > 0.05).During the entire 60-min experimental duration in the standing condition, FCR H-reflex amplitudes and M-waves were not significantly modulated  and the limb from which the H-reflex was recorded  of a sigmoidal function. Long-lasting H-reflex suppression was not significantly seen after the 5 min stepping task in our condition. In other words, it is possible that 5 min of stepping-related afferent feedback was insufficient to induce long-lasting suppression of the monosynaptic reflex circuit in the cervical cord.We found that the duration of the passive stepping strongly correlated to H-reflex suppression after the termination of stepping and decayed according to a slope parameter , were dependent on the duration of the stimulation  was partially supported by the MEXT/JSPS KAKENHI .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The NikR protein is a Ni2+-dependent transcriptional repressor of the nickel-ion uptake system in Escherichia coli, but its mutant protein (NikRm) is able to selectively bind uranyl ions in the interface of the two monomers. In this study, NikRm protein with ability to adsorb uranyl ions was displayed on the cell surface of Saccharomyces cerevisiae. To perform the binding of metal ions in the interface of the two monomers, two metal-binding domains (MBDs) of NikRm were tandemly fused via linker peptides and displayed on the yeast cell surface by fusion with the cell wall-anchoring domain of yeast α-agglutinin. The NikRm-MBD-displaying yeast cells with particular linker lengths showed the enhanced adsorption of uranyl ions in comparison to the control strain. By treating cells with citrate buffer (pH 4.3), the uranyl ions adsorbed on the cell surface were recovered. Our results indicate that the adsorption system by yeast cells displaying tandemly fused MBDs of NikRm is effective for simple and concentrated recovery of uranyl ions, as well as adsorption of uranyl ions.Uranium is one of the most important metal resources, and the technology for the recovery of uranyl ions (UO Uranium, an element from the actinide series, is one of the heaviest elements naturally occurring on earth, and is one of the most important metal resources used as nuclear fuel. The amount of minable uranium in the earth’s crust is estimated to be 5.5 million tons. Compared to other minor metals, uranium deposits are relatively widespread, with major world producers including Australia (23%), Kazakhstan (15%), Russia (10%), and others. Based on annual use, however, uranium is predicted to be depleted within 100 years. It is therefore important to develop novel uranium sources to maintain a stable energy supply. Seawater contains a considerable amount of uranium, and the amount estimated to be present in the oceans (4.6 billion tons) is comparable to 20 thousand years’ worth of usage. Therefore, the development of a uranium-recovering system from seawater could provide a virtually permanent supply of energy. However, it is difficult to recover uranium from seawater using existing techniques due to the low concentration of uranyl ions  in seawater.For the recovery of metal ions from a solution containing metal ions at low concentrations, the bioadsorption of metal ions using microorganisms is an attractive technology. Notably, cell-surface adsorption by yeast cell surface engineering, which enables the display of functional proteins by fusion with cell wall-anchoring proteins, is an advantageous strategy ,2,3,4,5.22+) and its complexes, is the most stable under aerobic and aqueous conditions. Serum proteins including transferrin  was used as a host cell for recombinant DNA manipulation. Saccharomyces cerevisiae BY4741/Δsed1  was used as the host cell for the efficient display of proteins on the cell surface [E. coli was grown in Luria-Bertani medium (1% (w/v) tryptone, 0.5% yeast extract, 0.5% sodium chloride) containing 100 μg/mL ampicillin. Yeast cells were precultivated aerobically in synthetic dextrose medium (SDC + HML) (0.67% (w/v) yeast nitrogen base without amino acids, 2% glucose, 0.5% casamino acids, 0.002% l-histidine, 0.002% l-methionine, and 0.003% l-leucine) buffered by 100 mM 2-morpholinoethanesulfonic acid (MES) at pH 6.8, and then cultivated in the same medium. surface . E. coliC-terminal 85 amino acids of NikR including MBD was amplified from E. coli DH5α genomic DNA by PCR using primers NikR-MBD-F and NikR-MBD-R. The amplified DNA fragment was digested with Bgl II and Xho I, and then inserted into the Bgl II-Xho I section of the pULD1 plasmid [Bgl II and Sal I cohesive termini  were amplified by PCR using the forward primer Bgl II-Sal I-NikRm-F and reverse primers NikRm-L0-R, NikRm-L3-R, NikRm-L6-R, NikRm-L9-R, or NikRm-L12-R, respectively. Amplified DNA fragments were digested with Bgl II and Xho I, and inserted into the Bgl II-Sal I section of pULD1-mL3 or pULD1-mL6 . The constructed plasmids for the cell surface display of the tandemly fused MBDs of NikRm were named pULD1-mLxLy . The plasmid pULD1-s [strep-tag and pULD1 for the cell surface display of the FLAG-tag were used as negative controls of immunofluorescence labeling and bioadsorption experiments, respectively.All primers used in the plasmid construction are listed in  plasmid  for the  termini . The DNA pULD1-s  for the S. cerevisiae cells. Transformants were isolated by incubation on a plate of SDC + HML medium at 30 °C for 48 h. All constructed yeast strains are listed in The lithium acetate method  was usedTo confirm the display of tandemly fused MBDs of NikRm on the yeast cell surface, immunofluorescent labeling of cells was performed using the FLAG epitope tag as described previously . Cells wg, and cell densities were adjusted to an optical density of 3.0 at 600 nm (OD600) with PBS (pH 7.4). Fluorescence intensity was measured using a Fluoroskan Ascent FL fluorometer  in a tissue-culture plate  using excitation and emission wavelengths of 485 nm and 527 nm respectively. The measured values of fluorescence intensity correspond to the number of the displayed protein on the cell surface.To estimate the number of tandemly fused MBDs of NikRm that were displayed on the yeast cell surface, the fluorescence intensity of cells was evaluated following immunofluorescent labeling. The immunofluorescence-labeled cells were precipitated by centrifugation at 800× g for 10 min. The cells were incubated in a metal solution containing 100 ppb of Be, Bi, Co, In, and U that was prepared by diluting 1 mg/L custom assurance standard  with ultrapure water to an OD600 of 0.25. The adsorption reaction was performed at room temperature with rotary shaking and centrifuged at 800× g for 10 min. The number of residual uranyl ions in the supernatant was quantified by ICP-MS . For adsorption, the pH of the metal solution was adjusted to 4.0 with HNO3 or NaOH. The resulting cell pellets were treated with ultrapure water, MES buffer, or citrate buffer as release solutions to recover cell-surface-bound uranyl ions. A mixture of cells and release solution was incubated at room temperature with rotary shaking followed by centrifugation at 800× g for 10 min. The concentration of uranyl ions released into the resulting supernatant was also measured by ICP-MS. The adsorption efficiency and the recovery efficiency were calculated as follows :The bioadsorption of metal ions by the cell surface-engineered yeasts was measured according to the previously describe protocol . Prior tThe results of our current study suggest that cell-surface-engineered yeast cells displaying tandemly fused MBDs of NikRm are effective in the adsorption of uranyl ions, which are the oxyanion form of uranium present in seawater. Our adsorption technique is important to the future of stable energy supply, since uranium is used as nuclear fuel. The development of cell-surface adsorption systems by yeast cell surface engineering would be advantageous for the adsorption and recovery of various metal ions, and may contribute to the effective recycling of valuable metal ions from metal-containing water in industrial applications as well as in natural environments.


To study the in vivo and in vitro effects of adding oxygen carbon nanotubes (CNTs) to chemotherapy for breast cancer.MCF-7 and SK-BR-3 breast cancer cells were co-cultured with paclitaxel and then exposed to oxygen-CNTs under hypoxic conditions. Cell proliferation, viability, and apoptosis rate were analyzed. Hypoxia-inducible factor-1 alpha (HIF-1α) expression was measured using reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Nude mice were used as a human breast cancer model to explore the impact of oxygen-CNTs on the in vivo chemotherapeutic effect of paclitaxel.Oxygen-CNTs had no significant effects on the growth of breast cancer cells under normoxia and hypoxia. However, in the hypoxic environment, oxygen-CNTs significantly enhanced the inhibitory effect of paclitaxel on cell proliferation, as well as the apoptosis rate. Under hypoxia, downregulation of HIF-1α and upregulation of caspase-3, caspase-8, caspase-9, LC3 and Beclin-1 were observed when paclitaxel was combined with oxygen-CNT. Furthermore, addition of oxygen-CNTs to chemotherapy was found to significantly reduce tumor weight in the tumor-bearing mice model.Oxygen-CNTs can significantly increase the chemotherapeutic effect of paclitaxel on breast cancer cells. Oxygen-CNTs may be a potential chemosensitizer in breast cancer therapy. Breast cancer is one of the most common cancers in women, affecting 1.2 million women in the world and being responsible for the death of 500,000 women each year. China is among the countries with the fastest growing incidence of breast cancer, and statistical data from the Chinese Anti-Cancer Association show that in recent years, the incidence of breast cancer has been increasing by 3% every year, and has been accompanied by a decrease in the age of onset Despite great advances in breast cancer treatment, there are still cases of tumor resistance to treatments. Hypoxia is an important factor that affects the efficacy of treatment. Indeed, hypoxia significantly increases tumor resistance to chemotherapy and radiotherapy, and may also increase the invasiveness of the tumor In recent years, nanotechnology has been used for a wide range of applications in medicine and biology. Carbon nanotubes (CNTs) are expected to achieve a breakthrough in the treatment of breast cancer and other tumors by reducing the tumors' resistance to radiotherapy and chemotherapy We undertook the present study to test the hypothesis that oxygen-CNTs inhibit breast cancer cell proliferation induced by HIF1α. Our findings show that oxygen-CNTs are potent chemosensitizers that improve the effects of paclitaxel in vivo and in vitro. Our results further indicate that oxygen-CNTs induce this effect by improving the hypoxia status and sensitivity to chemotherapy.Functional oxygen single-walled carbon nanotubes (SWCNTs) were provided by the Mechanical and Power Engineering College of Shanghai Jiaotong University . DMEM, RPMI 1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were from GIBCO .Nude mice were used as an animal model to study the biological effects of oxygen-CNTs. This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Xinhua Hospital, affiliated to Shanghai Jiaotong University School of Medicine, China. Surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.CNTs were purified by acid treatment and high energy ball mill; they were of >95% purity, 0.5–2 µm in length, and 10–20 nm in diameter. Single-wall carbon nanotubes (1 g)  were ground in an agate mortar for 1 h. Then, 0.1 g of the ground CNT powder was placed in a 500-mL Erlenmeyer containing 50 mL of an acid mixture (3∶1 v/v ratio of 98% sulfuric acid and 78% nitric acid), for a concentration of 2 mg of nanotubes per 1 ml of acid solution. The solution was treated in an oscillating ultrasonic bath  for 2 h at room temperature. Oxidative reflux was conducted for 0.5 h at 100°C, and then the reaction liquid were naturally cooled to room temperature, after which 300 mL of deionized water was added. The flask was kept still for some time until the precipitate containing oxygen-CNTs had deposited at the bottom of the flask. The supernatant was discarded, and the turbid liquid that remained was filtered through a millipore filter. The filter paper containing the oxygen-CNT residue was placed in a beaker containing deionized water, and was ultrasonicated  to separate the CNTs. The solution was poured into an Erlenmeyer and repeatedly washed, filtered and resuspended until the pH value was neutral. The oxygen-CNT-containing solution was then dried in an oven at 60°C. We obtained a black powder that was used for the further experiments.The constituent elements  of the oxygen CNTs were detected by X-ray photoelectron spectroscopy (XPS) analysis.Carbon nanotubes were dispersed, dropped on a copper screen and observed under a JEM-2010 high-resolution transmission electron microscope (TEM) to assess their distribution in solution.2) for 12h and then in a hypoxic incubator  for 48 h. Adherent cells were picked up from the culture and subjected to 0.25% trypsin digestion. Cells in the logarithmic growth phase were used for subsequent experiments.The breast cancer cell lines MCF-7 and SK-BR-3 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences . Cells were cultured in DMEM and RPMI1640 complete media . The cultures were incubated in a normoxic incubator  was used to determine cell viability. To conduct the assay, 10 µL of WST-1 (5 mg/mL) working solution was added to each well at a final concentration of 500 µg/mL. After the mixture in each well was incubated for 2.5 h, the absorbance was read with a microplate reader  at 450 nm. Relative cell viability was expressed as the percentage of viable cells divided by the number of viable cells in the control wells that were not treated with functional CNTs. The inhibition rate was calculated using the formula: cell inhibition rate = [1−absorbance of the experimental group/control group absorbance]×100%.5/mL and incubated at 37°C in an atmosphere containing 5% CO2, 1% O2, and 94% N2, for 24 h. The cells were then harvested by trypsinization and washed once with phosphate-buffered saline . After centrifugation, the cells were resuspended in 100 µL of binding buffer, and then stained with FITC-Annexin V and propidium iodide  for analysis of cell apoptosis. The reaction was developed under dark conditions for 10 min, after which 400 µL of binding buffer was added to the wells. Cells were counted using a flow cytometer  (channel selection FL1 and FL2).Cells in the logarithmic growth phase were seeded in 6-well plates at a density of 105′-ACC ACA GTC CAT GCC ATC AC-3′ (sense) and 5′-CCA CCA CCC TGT TGC TGT A-3′ (antisense) (471 bp). The primers for Ki67 were 5′-ATC GTC CCA GGT GGA AGA GTT-3′ (sense) and 5′-ATA GTA ACC AGG CGT CTC GTG G-3′ (antisense) (126 bp). The primers for survivin were 5′-AGC CCT TTC TCA AGG ACC AC-3′ (sense) and 5′-GCA CTT TCT TCG CAG TTT CC-3′ (antisense) (363 bp). PCR conditions were: 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The products were detected using 1.5–2% agarose gel  electrophoresis. Results were analyzed using the Bio-rad Gel DocTM XR+ imaging system .After treatment with paclitaxel and oxygen-CNTs for 48 h, cells were harvested. Total RNA was extracted using Trizol , and cDNA was produced by reverse transcription and amplification using the reverse transcription-polymerase chain reaction kit . The primers were synthesized by Sangon Co. . The primers for GADPH were Cells were washed and then suspended in 100 µL of lysate . The total protein concentration was determined using the bicinchoninic acid (BCA) assay and a microplate reader at 562 nm. After an equal amount of protein was loaded in each lane, the proteins were separated using 10% SDS w/v polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking the membrane with 5% (w/v) skim milk, target proteins were immunodetected using specific antibodies. All primary antibodies were used in a 1∶1000 dilution and incubated overnight at 4°C. After washing with Tris-buffered saline with Ttween-20 (TBST) three times for 20 min each, horseradish peroxidase (HRP)-conjugated antibody  was added as the secondary antibody. Positive bands were detected using ECL Plus Western Blotting Detection Regents. The results were scanned using the Bio-Rad Chemi Doc™ XRS+imaging system . Caspase-3, caspase-8, and caspase-9 mouse monoclonal antibodies and p27 rabbit monoclonal antibodies were purchased from Santa Cruz . β-Actin, LC3, and Beclin-1 rabbit monoclonal antibodies were purchased from Cell Signaling Technology .6 cells, twice a week). After the tumors were allowed to develop for 2 weeks, the animals were randomly grouped into one of three groups (n = 6/group). The animals in the vehicle group were administered an intraperitoneal injection of 0.9% normal saline. The animals in the paclitaxel group were intraperitoneally administered 20 µg/kg body weight paclitaxel each week. Finally, the animals in the oxygen-CNT group were intraperitoneally administered 5 mg/kg body weight oxygen-CNTs each week local injection with lots of dot around the tumor. At the end of the experiment, the animals were euthanized with a CO2 overdose and decapitated. Tumor tissue from each animal was excised and then weighed after the excess normal tissue was trimmed off. The inhibition rate was calculated using the formula inhibition rate = [/Weightcontrol group]×100%.Six-week-old female Balb/c nu/nu mice were obtained from the Shanghai Animal Center . The animals were housed in sterilized cages with filtered air, 12-h light-dark cycle, and had free access to sterile water and food. After 1 week of acclimatization, breast cancer cells were injected into the right flanks of each mouse . A P-value<0.05 was considered statistically significant.TEM pictures of functional oxygen single-walled carbon nanotubes after oxygen modification showed that the carbon nanotubes were evenly dispersed in solution .The WST-1 assay showed that the oxygen-CNTs had no influence on the activity of MCF-7 cells in the normoxic environment (OD <0.2) . MoreoveThe cell apoptosis rates determined using flow cytometry are shown in RT-PCR showed that survivin and Ki67 mRNA expression were significantly reduced by 30–45% with the addition of oxygen-CNTs compared with paclitaxel only P<0.05, . Under hThe expression of the apoptosis-related proteins caspase-3, caspase-8 and caspase-9, and of the autophagy-associated proteins LC3 and Beclin-1 was increased after 48 h of treatment, and the expression of these proteins further increased with the addition of oxygen-CNTs  .in vivo and in vitro effects of adding oxygen-CNTs to chemotherapy for hypoxic breast cancer. Results showed that in the normoxic and hypoxic environments, oxygen-CNTs had no significant effects on the growth of breast cancer cells. However, under hypoxia, oxygen-CNTs significantly enhanced the inhibitory effect of paclitaxel on cell proliferation, as well as the apoptosis rate. Under hypoxia, downregulation of HIF-1α and upregulation of caspase-3, caspase-8, caspase-9, LC3 and Beclin-1 were observed when paclitaxel was combined with oxygen-CNT. Furthermore, addition of oxygen-CNTs to chemotherapy was found to significantly reduce tumor weight in the tumor-bearing mice model.The aim of the present study was to assess the CNTs present a high surface area, excellent chemical stability and are able to adsorb and conjugate with many chemical elements or macromolecules. Furthermore, they are able to enter into cells and to keep the bound molecules intact during transport Since tumors grow rapidly, the proliferation rate of tumor cells is higher than that of cells in the surrounding blood vessels, leading to severe local tissue hypoxia, which triggers the tumor cells into a series of stress responses HIF-1 plays a key regulatory role in angiogenesis; it is associated with tumor development, invasion and metastasis, and affects the prognosis of most malignant tumors In the present study, under both normoxic and hypoxic conditions, oxygen-CNTs alone did not have an effect on the growth of breast cancer cells. However, oxygen-CNTs combined with paclitaxel showed more pronounced inhibition of the activity of breast cancer cells than paclitaxel alone under hypoxic conditions. These results are in agreement with previous studies that showed that the reoxygenation of hypoxic cancer cells led to better responses to paclitaxel treatment Therefore, it is possible that the addition of oxygen-CNTs under hypoxic conditions increased the availability of oxygen to the cells, increasing their sensitivity to paclitaxel and subsequently increasing apoptosis and autophagy. However, the specific mechanism by which oxygen-CNTs improve the chemotherapeutic effect of anti-cancer drugs needs to be further studied. The addition of oxygen to the cells did not seem to be toxic by itself. Therefore, we assume that the effects we observed were due to the chemo-sensitizing effects via decreased HIF-1α expression.in vitro and in vivo, and significantly improved the chemotherapeutic effect of paclitaxel. Oxygen-CNTs may therefore be a potential chemotherapy sensitizer for breast cancer treatment.Oxygen-CNTs improved the hypoxia status of breast cancer cells


Vibrio isolates of various serogroups. These are valuable data for use in studying Vibrio cholerae genetic diversity, epidemic potential, and strain attribution.We present draft genome sequences of 15 clinical  Vibrio cholerae is a Gram-negative bacterium and the etiologic agent of cholera. Cholera is endemic in regions with poor sanitation and can be a significant problem wherever local infrastructure is disrupted due to natural or human-caused disasters. A recent bulletin of the World Health Organization reported that there are an estimated 2.8 million cases and 91,000 deaths annually due to cholera, with the highest burden being in children under the age of 5 years . 454 Titanium libraries were prepared and emulsion PCR (emPCR) was performed per the manufacturer’s instructions, prior to multiplexed sequencing and V. cholerae virulence factors are present in a subset of these genomes. For instance, we identified sequences corresponding to all or a large part of V. cholerae pathogenicity island 1 (VPI-1), not only in NIH41 and 63-93 , but also in nonepidemic strains 981-75, 1421-77, 571-88, 523-80, and 234-93. We identified sequences with homology to the cholera toxin genes in NIH-41 and 63-93, as expected, as well as in 571-88, 234-93, and 490-93. Finally, we identified sequences corresponding to all or part of VPI-2 in NIH-41 and 981-75. Given that these are draft genomes and in some cases gaps exist within these particular regions, further investigation is required to elucidate whether these virulence factors are functional. These draft genomes have the potential to aid our understanding of virulence and epidemicity in V. cholerae. These data contribute to broadening the reference genome collection available for studies aimed at V. cholerae genetic diversity and strain attribution.Preliminary analyses indicate that some of the major The sequence data were deposited in GenBank under project accession numbers listed in


The role of HLA-DR antigens in the clinicopathological features of autoimmune hepatitis (AIH) is not clearly understood. We examined the implications of HLA-DR antigens in Japanese AIH, including the effect of HLA-DR4 on the age and pattern of AIH onset, clinicopathological features, and treatment efficacy.A total of 132 AIH patients consecutively diagnosed and treated in 2000–2014 at 2 major hepatology centers of eastern Tokyo district were the subjects of this study. The frequency of HLA-DR phenotypes was compared with that in the healthy Japanese population. AIH patients were divided into HLA-DR4–positive or HLA-DR4–negative groups and further sub-classified into elderly and young-to-middle-aged groups, and differences in clinical and histological features were examined. Clinical features associated with the response to immunosuppressive therapy were also determined.P < 0.001), and the relative risk was 2.14 . HLA-DR4–positive AIH patients were younger than HLA-DR4–negative patients (P = 0.034). Serum IgG and IgM levels were higher  in HLA-DR4–positive patients. These differences were more prominent in elderly AIH patients. However, there was no difference in IgG and IgM levels between HLA-DR4–positive and HLA-DR4–negative patients of the young-to-middle-aged group. There were no differences in the histological features. In patients with refractory to immunosuppressive therapy, higher total bilirubin, longer prothrombin time, lower serum albumin, and lower platelet count were found. Imaging revealed splenomegaly to be more frequent in refractory patients than in non-refractory patients . HLA-DR phenotype distribution was similar regardless of response to immunosuppressive therapy.The frequency of the HLA-DR4 phenotype was significantly higher in AIH than in control subjects (59.7 % vs. 41.8 %, HLA-DR4 was the only DR antigen significantly associated with Japanese AIH. The clinical features of HLA-DR4–positive AIH differed between elderly patients and young-to-middle-aged patients. Treatment response depended on the severity of liver dysfunction but not on HLA-DR antigens. Autoimmune hepatitis (AIH) is a rare inflammatory liver disease, with prevalence rates of 5–20 per 100,000 in Europe and North America , 4. The As regards the immunogenetic background of AIH, HLA-DR3 (recently split into DR17 and DR18) and HLA-DR4 are associated with type 1 AIH . In JapaIn a report on North American patients, the clinical features of HLA-DR4–positive AIH differed from those of HLA-DR4–negative patients . In addiIn the present study, we thoroughly examined the role of HLA-DR antigens in Japanese AIH, including how HLA-DR4 influences the age of AIH onset and its clinical features. The association of HLA-DR antigens with the treatment efficacy was also examined.A total of 132 patients who had been consecutively diagnosed with AIH, treated, and examined for the HLA-DR antigen at Tokyo Metropolitan Bokutoh Hospital and the Jikei University School of Medicine Katsushika Medical Center (2 of the major hepatology centers in eastern Tokyo district) from the beginning of 2000 till May 2014 were the subjects of this study. AIH diagnosis was based on the Diagnostic Criteria of the International Autoimmune Hepatitis Group (IAHG) [The medical records of the subjects at the time of diagnosis were collected and analyzed retrospectively. In addition, the clinical course of all AIH patients was surveyed. The collected laboratory data included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TB), albumin (Alb), platelet count (PLT), prothrombin time (PT), immunoglobulin G (IgG), immunoglobulin M (IgM), anti-nuclear antibodies (ANA), anti-mitochondrial antibodies (AMA), HCV- and HBV-related viral markers, drug history, average alcohol intake, liver histology, other concomitant autoimmune diseases, and other defined autoantibodies. ANA and AMA were detected and titrated by standard indirect immunofluorescence. For imaging, computed tomography and/or ultrasonography were performed for all but 4 patients, and the presence or absence of splenomegaly was evaluated by radiologists. HLA-DR antigens were assayed in all patients by PCR-based reverse sequence specific oligonucleotide typing . Pre-treatment AIH score was calculated in every patient. Then, the frequencies of HLA-DR phenotypes were assessed, and the importance of HLA-DR4 in clinical features in elderly or younger AIH patients was analyzed. The effect of HLA-DR antigens on response to immunosuppressive therapy was also examined.The protocol of this study was approved by the Ethical committee of Tokyo Metropolitan Bokutoh Hospital and the Ethical committee of the Jikei University School of Medicine. Information of the protocol of this study was explained to the participants, and verbal informed consent was obtained from all patients. This study complied with the standards of the 2008 Declaration of Helsinki and current ethical guidelines.AIH patients were divided into HLA-DR4–positive and HLA-DR4–negative groups. They were further sub-classified into an elderly group  and a young-to-middle-aged group . In each age group, the differences in the clinical features between HLA-DR4 − positive and HLA-DR4 − negative patients were examined.Sufficient liver tissue  was obtained by percutaneous liver biopsy before starting therapy in 116 out of 132 AIH patients. Among the remaining 16 patients, consent for liver biopsy could not be obtained in 2; immunosuppressive drugs had already been taken based on the clinical diagnosis of AIH in 3; and percutaneous liver biopsy was contraindicated because of severe liver damage in 8 patients. Biopsy specimens could not be obtained in 2 patients, and sufficient length of biopsy specimen was not achieved in 1 patient. The biopsy samples were stained by hematoxylin/eosin and Masson’s trichrome or Azan. The pathological features of AIH were determined collectively by a single pathologist (AS) blinded to clinical information. Pathological scoring of AIH based on the degree of interface hepatitis, predominance of lymphoplasmacytic infiltrate, and presence of rosetting of liver cells was performed according to the diagnostic criteria of the IAHG [Patients with acute liver dysfunction  or with acute liver-related symptoms  without evidence of liver dysfunction in the past (more than 6 months before diagnosis) was defined as acute-onset AIH, while the others were defined as chronic onset AIH. This classification was based on a report by Miyake et al. .HLA-DR phenotype frequencies in AIH were determined and compared with those in a large-scale population study on healthy Japanese people by the HLA Laboratory, Kyoto, Japan .Of 132 AIH patients, 121 (92 %) were treated by prednisolone alone or in combination with an immunomodulator . The remaining 11 patients were treated with ursodeoxycholic acid alone because of mild inflammatory activity . PrednisU-test was used to analyze differences between continuous variables. Statistical significance was determined by a two-tailed test and P-values of ≤0.05 were considered significant. All statistical analyses were carried out using STATISTICA for Windows version 6 .Results are expressed as number (%) or median (minimum–maximum). Fisher’s exact test or chi-square test was used to analyze differences in categorical data. The Mann–Whitney The frequency of the HLA-DR4 phenotype was significantly higher in AIH than in control individuals . The relative risk (RR) of HLA-DR4 was 2.14 .Among the other HLA-DR antigens, HLA-DR14 tended to be more frequent and HLA-DR11, HLA-DR12, and HLA-DR15 less frequent in AIH compared with control subjects, although the differences were not statistically significant , 22 patients had HLA-DR4, while 27 did not. Both serum IgG and IgM levels were higher in elderly HLA-DR4–positive AIH patients than in HLA-DR4–negative patients. These differences between HLA-DR4–positive and HLA-DR4–negative elderly AIH patients were greater than those shown with the whole population. Moreover, pretreatment AIH scores  were significantly higher in the HLA-DR4–positive group than in the HLA-DR4–negative group. Advanced fibrosis tended to be more frequent in HLA-DR4–positive elderly AIH patients Table . With reIn AIH patients younger than 55 years (young-to-middle-aged AIH), 34 of 49 (63.8 %) patients had HLA-DR4. The clinicopathological features between HLA-DR4–positive and HLA-DR4–negative AIH were similar except for splenomegaly, which was less frequently found in HLA-DR4–positive patients. In contrast to elderly AIH, serum levels of IgG and IgM or pretreatment AIH scores  did not differ between HLA-DR4–positive and HLA-DR4–negative patients. Moreover, the fibrosis stage was similar between HLA-DR4–positive and HLA-DR4–negative AIH . In the refractory group, the 6 patients who did not undergo liver biopsy were those for whom liver biopsy was contraindicated because of severe hepatic deterioration (Table The acute-onset AIH was more frequent in the patients in whom remission was induced by the standard therapy within 6 months (non-refractory group) than in the patients resistant to the standard therapy (refractory group). Splenomegaly was less frequently found in the non-refractory group than in the refractory group. TB was lower, PT was longer, and Alb and PLT were higher in the non-refractory group. Moreover, the serum ALP level was lower in the non-refractory group. The occurrence of advanced liver fibrosis was not different, but the number of patients who did not undergo liver biopsy was higher in the refractory group than in the non-refractory group  , warrantWe next examined the role of HLA-DR4 on the clinical features of type 1 AIH. HLA-DR4–positive AIH patients were young and had hypergammaglobulinemia without decreases in albumin levels or platelet count. Marked hypergammaglobulinemia might be a common feature of HLA-DR4–positive AIH in Japan . AccordiInterestingly, the incidence of HLA-DR4 differed between elderly and young-to-middle-aged patients. The rate of HLA-DR4 in elderly patients was equal to that in normal subjects. Therefore, HLA-DR4 is not necessarily a risk factor contributing to the onset of AIH in elderly individuals. However, in North America, the frequency of DR4-positive AIH has been increasing in elderly patients, with a decline of DR3-positive AIH . MeanwhiA critical difference between the present study and the earlier one might be the high proportion of elderly AIH patients in our study. Only 20 % of the patients were elderly in the past study, while 34.1 % of the patients in the current study were elderly. This can be attributed to the increase in older population in Japan and recent progress in the proper diagnosis of AIH onset in the elderly. In addition, when the results were compared with those of Caucasian type 1 AIH, HLA-DR3–positive AIH was negligible in the Japanese population.HLA-DR4–positive elderly AIH patients exhibited prominent hyperglobulinemia, which is one of the characteristic features of typical AIH. In contrast, HLA-DR4–negative elderly AIH patients may exhibit atypical clinical results. Therefore, it is necessary to diagnose HLA-DR4–negative elderly AIH patients carefully.In young-to-middle-aged AIH patients, the rate of HLA-DR4 was higher. The clinical characteristics were not different between HLA-DR4–positive and HLA-DR4–negative AIH. However, the number of AIH patients younger than 30 years was only 7 in our study. Therefore, precise characterization of AIH in young adults and/or children requires further research. For children or young adults, careful diagnosis of AIH by an experienced hepatologist is essential because differential diagnosis from other causes of liver damage, including chronic active Epstein–Barr virus infection, is difficult .Finally, we examined the contribution of the HLA-DR antigen to the response to immunosuppressive therapy. Association of HLA-DR14 with a favorable response to corticosteroid therapy , better Patients with refractory AIH had severe deterioration of liver function. Therefore, we assumed that treatment efficacy was largely influenced by the functional deterioration of the liver at the time of starting therapy. Thus, proper diagnosis and therapy without delay are key factors for the successful treatment of AIH. Meanwhile, the effect of HLA-DR on treatment efficacy may be negligible; however, this cannot be conclusive because of the limited patient number in our study.The HLA-DR4 antigen alone was associated with Japanese AIH. No other predisposing HLA-DR antigen was recognized in this study. The impact of HLA-DR4 on the features of AIH differed between elderly patients and younger patients. Treatment response was associated with the severity of liver disease but not with HLA-DR antigen. AIH refractory to immunosuppressive therapy was diagnosed in cases of severe liver dysfunction. Early recognition, proper diagnosis, and immediate start of immunosuppressive therapy are essential for favorable treatment outcome.